Methods of inhibiting the expression of genes through short single-stranded oligonucleotides or oligoribonucleotides or modified oligonucleotides perfectly complementary to the target mRNA are known as “antisense”. The use of antisense oligonucleotides (ASOs) as a tool to help elucidate gene function is well-described. Antisense oligonucleotides are also being evaluated as medicaments for a wide variety of diseases.
As an alternative to antisense, sequence-specific degradation of mRNAs with oligonucleotides can also be triggered by short RNA duplexes by an RNA interference (RNAi) mechanism. RNA interference is a process of sequence-specific, post-transcriptional gene silencing initiated by double-stranded RNA that is homologous in sequence to the silenced gene. The modulation of the function of a target nucleic acid by oligoribonucleotides which inhibit the expression of said target nucleic acid is generally referred to as “RNAi” or “RNA interference”. Effective target-gene specific inhibition is usually achieved by short double-stranded (ds) oligoribonucleotide and with an overhang of approximately 2 nucleotides at the ends of at least 1 strand of the duplex. Such double-stranded oligoribonucleotides are known as short interfering RNAs (siRNAs) and have for instance been used as a tools to help elucidate gene function.
Great efforts are being made to develop oligonucleotides inhibiting the expression of specific target gene for therapeutic uses. One of the problems encountered is that, due to the special characteristics of oligonucleotides (such as for example high molecular weight, high amounts of negative charge, metabolic instability), delivery of free oligonucleotides to target tissues is generally much more limited in terms of the variety of disease target tissues, than for small molecule inhibitors: for instance, free oligonucleotides have low bioavailability when given orally to patients, systemic delivery of oligonucleotides leads to high levels of drug concentrated in a small number of organs, for example in liver, spleen and kidney, where the distribution is dependent on the format of the oligonucleotide (Feng et al., in 2000, European Journal of Pharmaceutical Sciences 10, 179-186). Delivery of oligonucleotides to the Central Nervous System (CNS) poses particular problems due to the blood brain barrier (BBB) that free oligonucleotides cannot cross. One means to deliver oligonucleotides into the CNS is intrathecal delivery. However, the oligonucleotides need also to be efficiently internalised into target cells of the CNS in order to achieve the desired therapeutic effect. Usually, delivery reagents such as liposomes, cationic lipids, nanoparticles forming complexes are utilized in order to aid the intracellular internalization of oligonucleotides into cells of neuronal origin. However, it is of considerable economic and technical advantage in the development of drugs if the desired pharmacological effects can be achieved without the use of tissue delivery reagents. So far, the only report describing short dsRNAs entering mammalian cells without the aid of a delivery reagent show a poor effect (Milhaud, Pierre G. et al., J. Interferon Res. (1991), 11(5), 261-5). We have now surprisingly found in accordance with the present invention, that intrathecally delivered siRNAs efficiently enter CNS tissues and are efficiently internalized into cells of the CNS system. Thus, the present invention now provides for the first time a method for functional downregulation of target genes by dsRNA in the CNS in vivo, thereby affecting the disease phenotype, by delivering siRNA to the CNS.